Fig. 6
From: Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ
Generation of gene cassette knock-in mice by the enhanced PITCh system. a Schematic diagram of pronuclear injection of Cas9 protein, Actb and gRNA-s1 crRNAs, tracrRNA, PITCh-donor, and Exo1 mRNA. b PCR screenings of knock-in newborns. c Summary of Actb-TetO-FLEX-hM3Dq/mCherry knock-in mouse production by the enhanced PITCh system. d Sequences of boundaries between Actb and TetO-FLEX- hM3Dq/mCherry cassette. Blue and red characters indicate microhomologies and partial crRNA(gRNA-s1) target sequence, respectively. IF: internal forward primer, IR: internal reverse primer, LF: left forward primer, LR: left reverse primer, RF: right forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, WT: wildtype, KI: knock-in, and KI/+: tail genomic DNA of F1 heterozygous knock-in pup derived from #13 (KI#2) F0 knock-in mouse